The present invention relates to forced intercalation probes (FIT-probes) comprising at least one nucleoside analogue which comprises at least a fluorescent artificial nucleobase directly bound to a carbon of a modified sugar moiety wherein said modified sugar moiety is a carba-sugar or an amino acid nucleic acid (AANA). Thereby the nucleoside analogue is incorporated into DNA or RNA in the place of a single native base.
As such, FIT-probes may be employed in a large number of applications including genetic diagnostics, disease predisposition, pharmacogenetics and pathogen detection. The FIT-probes exhibit a simplistic mode of action and are able to detect single base alteration. They further possess few design constraints and show melting peak data which can be interpreted easily. The assay has been demonstrated to function efficiently directly from samples without prior purification of nucleic acids making the probe technology suitable for point-of-care diagnostics.
Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. SNPs are also thought to be key enablers in realizing the concept of personalized medicine. However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as with matched cohorts with and without a disease).
The polymerase chain reaction (PCR) by using hybridization probes is an extremely versatile technique for copying and qualitative and quantitative detection of DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. Diagnostics for genetic diseases are run and sequence analysis of DNA are carried out by hybridization of RNA transcripts with oligonucleotide array microchips.
A hybridization probe is a fragment of DNA or RNA of variable length (usually 100-1000 bases long), which is used to detect in DNA or RNA samples the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labelled probe is first denatured (by heating or under alkaline conditions) into single stranded DNA and then hybridized to the target DNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ.
DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via imaging techniques. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied—high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, and to which a mobile cDNA target is hybridized. Depending on the method the probe may be synthesized via phosphoamidite technology or generated and labelled by PCR amplification.
Fluorescence-labelled DNA probes play an important role in recent development of the detection of single-base alterations. The single-base discrimination in nearly all reported methods is achieved directly or indirectly in the basis of different hybridization efficiency between matched and mismatched target DNA/probe DNA duplexes. However, as far as the detection relies on the hybridization event, such DNA probes have inherent limitations in their selectivity. The differences in the hybridization efficiency vary with sequence context, and often are very small for the detection of a single-base mismatch in long-target DNA.
Methods having only one chromophore are preferred. Known methods are HyBeacon, LightUp-probes, base discriminating fluorosides (BDF) and FIT-probes.
HyBeacon probes provide a homogeneous method of ultrarapid sequence analysis that allows samples to be genotyped in a short time. These fluorescent probes are capable of reliably detecting specific DNA targets and discriminating closely related sequences, including those containing single nucleotide polymorphisms (SNPs). The design of the HyBeacon probe is characterized by a fluorescent derivative anchored on a nucleobase. The fluorescence of the chromophore is quenched in the unlinked state by batch with the nucleobase. In the case of hybridization with the target-DNA the quenching is decreased. However, selectivity of HyBeacon probes is limited.
The light-up probe is a recently developed probe for monitoring PCR amplification in real time. The design is characterized by anchoring of an intercalation dye at the end of an oligonucleotide over a flexible linker. It is a peptide nucleic acid (PNA) coupled to an asymmetric cyanine dye that becomes fluorescent upon binding nucleic acids. The light-up probe is used to monitor product accumulation in regular three steps PCR. It is designed to bind target DNA at annealing temperature, where the fluorescent signal is recorded, and to dissociate at elongation temperature. The differentiation of single mismatched complex is only possible by stringent hybridisation conditions.
A further strategy to discriminate single-base alterations are base-discriminating fluorescent (BDF) oligonucleotides probes. The BDF probes containing these fluorescent nucleosides selectively emit fluorescence only when the base opposite the BDF base is a target base. Oligonucleotides containing these BDF nucleosides act as effective reporter probes for homogeneous SNP typing of DNA samples. It is disadvantageous that the BDF probes are not universal usable or that they demonstrate the presence of a perfect complementary target-DNA by decreasing of the fluorescence.
Forced intercalation probes (FIT-probes) are peptide nucleic acid-based probes (PNA) in which the thiazole orange dye replaces a canonical nucleobase (WO 2006/072368A2). FIT-probes are used in homogenous DNA detection. The analysis is based on sequence-specific binding of the FIT-probe with DNA. FIG. 1A-shows the principle of detection. Binding of the FIT-probe places thiazole orange in the interior of the formed duplex. The intercalation of thiazole orange between nucleobases of the formed probe-target duplex restricts the torsional flexibility of the two heterocyclic ring systems (FIG. 1B). As a result, FIT probes show strong enhancements of fluorescence upon hybridization (FIG. 1A1). A less remarkable attenuation of fluorescence is observed when forcing thiazole orange to intercalate next to a mismatched base pair (FIG. 1A2). This base specificity of fluorescence signalling, which adds to the specificity of probe-target recognition, allows the detection of single base mutations even at non-stringent hybridization conditions. However, FIT-probes based on PNA show a high lipophilic character in comparison to DNA. Due to said differences PNA probes show undesirable high adhesion. Further, it is not possible to modify PNA chemically or with enzymes in the great diversity of DNA and the automated DNA synthesis and analysis is not compatible with the PNA systems.
Alternative FIT-probes which are based on the normal DNA sugar backbone are difficult to synthesize. To stabilize the fluorescent dye as base analogue in said probes long and flexible linker groups, such as substituted or unsubstituted alkyl chains, have to be included between the C1-atom of the sugar ring and the fluorescent dye. Furthermore, these probes show only a very weak signal intensity which is not sufficient to detect single base mutations effectively.
Thus, it is the object of the present invention to provide FIT-probes which show a strong fluorescence signal so that single base mutations can be detected reliably. It should be further possible to synthesize and analyze said FIT-probes by the automated systems usually used for DNA or RNA.